Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection.

Macrophage metabolic plasticity is central to inflammatory programming, yet mechanisms of coordinating metabolic and inflammatory programs during infection are poorly defined. Here, we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. We find that staggered Toll-like receptor and type I IFN signaling in macrophages permit a transient energetic state of combined oxidative phosphorylation (OXPHOS) and aerobic glycolysis followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. This disruption promotes type I IFN, suppressing other pro-inflammatory cytokines, notably interleukin-1ß. Upon infection, iNOS expression peaks at 24 h, followed by lactate-driven Nos2 repression via histone lactylation. Type I IFN pre-conditioning prolongs infection-induced iNOS expression, amplifying type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN results in elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Non-canonical activation of IRE1α during Candida albicans infection enhances macrophage fungicidal activity.

Infection by intracellular pathogens can trigger activation of the IRE1α branch of the unfolded protein response (UPR), which then modulates innate immunity and infection outcomes during bacterial or viral infection. However, the mechanisms by which infection activates IRE1α have not been fully elucidated. While recognition of microbe-associated molecular patterns can activate IRE1α, it is unclear whether this depends on the canonical role of IRE1α in detecting misfolded proteins. Here, we report that Candida albicans infection of macrophages results in IRE1α activation through C-type lectin receptor signaling, reinforcing a role for IRE1α as a central regulator of host responses to infection by a broad range of pathogens. However, IRE1α activation was not preceded by protein misfolding in response to either C. albicans infection or lipopolysaccharide treatment, implicating a non-canonical mode of IRE1α activation after recognition of microbial patterns. Investigation of the phenotypic consequences of IRE1α activation in macrophage antimicrobial responses revealed that IRE1α activity enhances the fungicidal activity of macrophages. Macrophages lacking IRE1α activity displayed inefficient phagolysosomal fusion, enabling C. albicans to evade fungal killing and escape the phagosome. Together, these data provide mechanistic insight for the non-canonical activation of IRE1α during infection, and reveal central roles for IRE1α in macrophage antifungal responses.

Prospective, randomized, double blind comparison of suture materials with and without triclosan in dogs undergoing tibial plateau leveling osteotomy.

Objectives: To determine if triclosan-impregnated suture decreases surgical site infection rates after tibial plateau leveling osteotomy (TPLO) in dogs. Sample population: There were 116 dogs with naturally occurring cranial cruciate ligament disease presenting for treatment with TPLO. Procedures: Written consent was obtained by all clients in order to be included in this study. Dogs were randomly assigned a suture type immediately before the start of anesthesia. Infection rates were compared between the suture groups, as were the gender, duration of anesthesia, duration of surgery, age of dog, weight, length of incision, and stifle side. Direct examination by a veterinarian was conducted at 24 h, 10 to 14 d, and 8 to 12 wk after surgery. If the dogs did not return for direct examination, owners were contacted by a veterinarian and phone interviews were conducted. Results: Overall, 12.9% of the incisions were diagnosed with a surgical site infection (SSI). The SSI rate for dogs that received the triclosan suture was 5.35% (3/56), and the rate for dogs that received the regular suture was 19.64% (11/56), with P = 0.016. The duration of anesthesia, duration of surgery, age, weight, length of incision, and right versus left stifle did not show a significant difference in infection rates. The suture type did have a significant effect, and triclosan-impregnated suture had a decreased infection rate when compared to regular suture. Gender also had a significant effect, with P = 0.032. Conclusion: Triclosan-impregnated suture decreased SSI when used for closure in dogs undergoing TPLO. Triclosan-impregnated suture may be considered a material of choice to close surgical wounds at risk of SSI when implants are used.

Comparaison prospective, randomisée, en double aveugle des matériaux de suture avec et sans triclosan chez les chiens subissant une ostéotomie de nivellement du plateau tibial. Objectifs: Déterminer si la suture imprégnée de triclosan diminue les taux d'infection du site opératoire après une ostéotomie de nivellement du plateau tibial (TPLO) chez le chien. Échantillon de population: Il y avait 116 chiens avec une pathologie naturelle du ligament croisé crânial se présentant pour un traitement avec TPLO. Procédures: Un consentement écrit a été obtenu par tous les clients afin d'être inclus dans cette étude. Les chiens ont été répartis au hasard à un type de suture immédiatement avant le début de l'anesthésie. Les taux d'infection ont été comparés entre les groupes de suture, de même que le sexe, la durée de l'anesthésie, la durée de la chirurgie, l'âge du chien, le poids, la longueur de l'incision et le côté du grasset. Un examen direct par un vétérinaire a été effectué à 24 h, 10 à 14 j et 8 à 12 semaines après la chirurgie. Si les chiens ne revenaient pas pour un examen direct, les propriétaires étaient contactés par un vétérinaire et des entretiens téléphoniques étaient menés. Résultats: Dans l'ensemble, 12,9 % des incisions ont été diagnostiquées avec une infection du site opératoire (SSI). Le taux de SSI pour les chiens ayant reçu la suture au triclosan était de 5,35 % (3/56) et le taux pour les chiens ayant reçu la suture régulière était de 19,64 % (11/56), avec P = 0,016. La durée de l'anesthésie, la durée de la chirurgie, l'âge, le poids, la longueur de l'incision et le grasset droit versus le gauche n'ont pas montré de différence significative dans les taux d'infection. Le type de suture avait un effet significatif et la suture imprégnée de triclosan avait un taux d'infection réduit par rapport à la suture ordinaire. Le sexe avait également un effet significatif, avec P = 0,032. Conclusion: La suture imprégnée de triclosan a diminué le SSI lorsqu'elle était utilisée pour la fermeture de l'incision chez les chiens subissant une TPLO. La suture imprégnée de triclosan peut être considérée comme un matériau de choix pour fermer les plaies chirurgicales à risque de SSI lorsque des implants sont utilisés.(Traduit par Dr Serge Messier).

Candida albicans selection for human commensalism results in substantial within-host diversity without decreasing fitness for invasive disease.

Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we examined 910 commensal isolates from 35 healthy donors to identify host niche-specific adaptations. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. We found that SC5314 was significantly different from the majority of both commensal and bloodstream isolates in its ability to induce host cell death. However, our commensal strains retained the capacity to cause disease in the Galleria model of systemic infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease.

FTO Suppresses STAT3 Activation and Modulates Proinflammatory Interferon-Stimulated Gene Expression.

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.

Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine.

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.

N6-Methyladenosine Regulates Host Responses to Viral Infection.

Recent discoveries have revealed that, during viral infection, the presence of the RNA modification N6-methyladenosine (m6A) on viral and cellular RNAs has profound impacts on infection outcome. Although m6A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m6A on viral and host RNAs remains a challenge for the field. m6A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m6A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m6A regulates viral infection.

Use of vessel sealing system for multiple partial lung lobectomies for spontaneous pneumothorax.

A wolf hybrid dog was presented for dyspnea and tachypnea. Thoracic radiographs revealed a pneumothorax. A median sternotomy was performed, and multiple pulmonary blebs were identified on several lung lobes. Multiple partial lung lobectomies were performed using a vessel sealing system. The dog was discharged 4 days after surgery free of clinical signs related to surgery or pneumothorax. This case represents a novel utilization of a vessel sealing system to remove the apex of the lung when there are numerous pulmonary lesions present. Key clinical message: A vessel sealing system simplified multiple partial lung lobectomies in an open thoracotomy. The system reduced tissue trauma as well as the amount of normal pulmonary tissue removed while efficiently creating a seal.

Utilisation d'un système de scellement des vaisseaux lors de lobectomies pulmonaires partielles pour un pneumothorax spontané. Un hybride chien-loup fut présenté pour dyspnée et tachypnée. Des radiographies thoraciques ont révélé un pneumothorax. Une sternotomie médiane fut effectuée et de multiples vésicules furent identifiés sur plusieurs lobes pulmonies. De multiples lobectomies partielles furent effectuées en utilisant un système de scellement des vaisseaux. Le chien obtint son congé 4 jours après la chirurgie sans aucun signe clinique relié à la chirurgie ou au pneumothorax. Ce cas représente une utilisation nouvelle d'un système de scellement des vaisseaux pour retirer l'apex des poumons lorsqu'il y a de nombreuses lésions pulmonaires présentes.Message clinique clé :Un système de scellement des vaisseaux simplifia des lobectomies pulmonaires partielles multiples lors d'une thoracotomie ouverte. Le système réduisit les traumatismes tissulaires ainsi que la quantité de tissu pulmonaire normal retirée tout en créant efficacement un sceau.(Traduit par Dr Serge Messier).

IL-27 signaling activates skin cells to induce innate antiviral proteins and protects against Zika virus infection.

In the skin, antiviral proteins and other immune molecules serve as the first line of innate antiviral defense. Here, we identify and characterize the induction of cutaneous innate antiviral proteins in response to IL-27 and its functional role during cutaneous defense against Zika virus infection. Transcriptional and phenotypic profiling of epidermal keratinocytes treated with IL-27 demonstrated activation of antiviral proteins OAS1, OAS2, OASL, and MX1 in the skin of both mice and humans. IL-27-mediated antiviral protein induction was found to occur in a STAT1- and IRF3-dependent but STAT2-independent manner. Moreover, using IL27ra mice, we demonstrate a significant role for IL-27 in inhibiting Zika virus morbidity and mortality following cutaneous, but not intravenous, inoculation. Together, our results demonstrate a critical and previously unrecognized role for IL-27 in cutaneous innate antiviral immunity against Zika virus.

The small GTPase RAB1B promotes antiviral innate immunity by interacting with TNF receptor-associated factor 3 (TRAF3).

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.

Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines.

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd-driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd-driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus).

Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): effects of gonadotropin-releasing hormone and human chorionic gonadotropin.

Research into the development of reproductive technologies for amphibians has increased in recent years due to the rapid decline of amphibian species globally. Reproductive technologies have great potential to overcome captive breeding failure and improve the propagation and genetic management of threatened species. However, the incorporation of these technologies into conservation breeding programs has been protracted, primarily as a result of trial-and-error approaches to the refinement of hormone therapies. The present study investigated the effects of: (1) GnRH-a dose (0, 0.5, 1, 2, 4, 8 or 16 µg g-1), and (2) hCG dose (0, 2.5, 5, 10, 20 or 40 IU g-1), on the sperm-release response of the critically endangered Booroolong frog. Administration of GnRH-a at a dose of 0.5 µg g-1 resulted in the greatest number of sperm released (mean total sperm = 3.5 ×106, n = 11). Overall, hCG was more effective at eliciting spermiation in Booroolong frogs, with peak sperm release (mean total sperm = 25.1 ×106, n = 10) occurring in response to a dose of 40 IU g-1. Sperm output in response to 40 IU g-1 hCG was greatest between 1 and 6 h and steadily declined between 8 and 24 h post-hormone administration. Percent sperm motility peaked between 4 and 10 h (58.1-62.7%), and sperm velocity between 4 and 12 h (24.3-27.2 µm s-1). Booroolong frogs join a small, but growing number of amphibian species that exhibit improved spermiation in response to hCG. Further research is required to identify optimal hormone-induction protocols for threatened amphibians and expedite the incorporation of reproductive technologies into CBPs.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog.

Carotenoids are known for their antioxidant capacity and are considered to play an important role in vertebrate growth and development. However, evidence for their beneficial effects remains limited, possibly because very few studies have tested for dose effects across different life stages. The present study investigated the effect of various doses of dietary beta-carotene supplements on the growth and development of larval and post-metamorphic Booroolong frogs (Litoria booroolongensis). Larval and post-metamorphic basal diets (containing 0.015 and 0.005 mg g-1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg g-1, 0.1 mg g-1, 1 mg g-1 and 10 mg g-1. Each treatment included 72 replicate individuals, and individuals remained on the same diet treatment over both life stages (spanning 53 experimental weeks). Our results show that larvae receiving an intermediate (1 mg g-1) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg g-1), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg g-1) on growth and development. However, juveniles fed the highest supplement dose (10 mg g-1) displayed significantly smaller body mass and lower body condition, compared to all other supplement doses, from 4-months through to sexual maturity (7-months). These findings indicate that beta-carotene supplementation has positive effects on growth and development, but only at intermediate doses, and only in the larval life stage. This knowledge may assist with amphibian conservation by expediting the rate that metamorphs can be generated in captive breeding programmes. More broadly, this is the first study to demonstrate both dose and life stage-dependent effects of dietary beta-carotene supplementation on vertebrate growth and development.

Hormone-induced spawning of the critically endangered northern corroboree frog Pseudophryne pengilleyi.

Fundamental knowledge of the optimal hormone concentrations required to stimulate amplexus and spawning in breeding pairs of amphibians is currently lacking, hindering our understanding of the proximate mechanisms underpinning mating behaviour. The present study investigated the effects of: (1) the dose of a gonadotropin-releasing hormone analogue (GnRH-a) administered; (2) male-female hormone administration interval; and (3) topical application of GnRH-a, on spawning success in the northern corroboree frog. Administration of GnRH-a at doses of 0.5, 1.0 and 2.0µgg-1 were highly successful, with a significantly greater proportion of hormone-treated pairs ovipositing (89-100%) compared with the 0µgg-1 treatment (22%). Of the hormone-treated pairs, those receiving 0.5µgg-1 GnRH-a exhibited the highest fertilisation success (61%). Administration of GnRH-a to males and females simultaneously (0h) was more effective than injecting males either 48 or 24h before the injection of females. Overall, administration of GnRH-a was highly successful at inducing spawning in northern corroboree frogs. For the first time, we also effectively induced spawning following the topical application of GnRH-a to the ventral pelvic region. Topical application of GnRH-a eliminates the need for specialised training in amphibian injection, and will allow assisted reproductive technologies to be adopted by a greater number of captive facilities globally.

A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

Survival, gene and metabolite responses of Litoria verreauxii alpina frogs to fungal disease chytridiomycosis.

The fungal skin disease chytridiomycosis has caused the devastating decline and extinction of hundreds of amphibian species globally, yet the potential for evolving resistance, and the underlying pathophysiological mechanisms remain poorly understood. We exposed 406 naïve, captive-raised alpine tree frogs (Litoria verreauxii alpina) from multiple populations (one evolutionarily naïve to chytridiomycosis) to the aetiological agent Batrachochytrium dendrobatidis in two concurrent and controlled infection experiments. We investigated (A) survival outcomes and clinical pathogen burdens between populations and clutches, and (B) individual host tissue responses to chytridiomycosis. Here we present multiple interrelated datasets associated with these exposure experiments, including animal signalment, survival and pathogen burden of 355 animals from Experiment A, and the following datasets related to 61 animals from Experiment B: animal signalment and pathogen burden; raw RNA-Seq reads from skin, liver and spleen tissues; de novo assembled transcriptomes for each tissue type; raw gene expression data; annotation data for each gene; and raw metabolite expression data from skin and liver tissues. These data provide an extensive baseline for future analyses.

Evolution of resistance to chytridiomycosis is associated with a robust early immune response.

Potentiating the evolution of immunity is a promising strategy for addressing biodiversity diseases. Assisted selection for infection resistance may enable the recovery and persistence of amphibians threatened by chytridiomycosis, a devastating fungal skin disease threatening hundreds of species globally. However, knowledge of the mechanisms involved in the natural evolution of immunity to chytridiomycosis is limited. Understanding the mechanisms of such resistance may help speed-assisted selection. Using a transcriptomics approach, we examined gene expression responses of endangered alpine tree frogs (Litoria verreauxii alpina) to subclinical infection, comparing two long-exposed populations with a naïve population. We performed a blinded, randomized and controlled exposure experiment, collecting skin, liver and spleen tissues at 4, 8 and 14 days postexposure from 51 wild-caught captively reared infection-naïve adult frogs for transcriptome assembly and differential gene expression analyses. We analysed our results in conjunction with infection intensity data, and the results of a large clinical survival experiment run concurrently with individuals from the same clutches. Here, we show that frogs from an evolutionarily long-exposed and phenotypically more resistant population of the highly susceptible alpine tree frog demonstrate a more robust innate and adaptive immune response at the critical early subclinical stage of infection when compared with two more susceptible populations. These results are consistent with the occurrence of evolution of resistance against chytridiomycosis, help to explain underlying resistance mechanisms, and provide genes of potential interest and sequence data for future research. We recommend further investigation of cell-mediated immunity pathways, the role of interferons and mechanisms of lymphocyte suppression.